In recent years, development of a producing process for efficient production of useful proteins such as pharmaceuticals has caught many interests. In this connection, there has been an ongoing development concerning use of plants not just as food but as factories for producing pharmaceuticals and other useful proteins. Such an effort is known as “molecular agriculture” and much is expected from this next-generation agriculture.
Currently, production of useful proteins in plants employs either a method using transgenic plants, or a method in which a plant is infected with a virus vector. (For details of the former, see Non-Patent Document 5: Transgenic plants as factories for biopharmaceuticals Glynis Giddings, Gordon Allison, Douglas Brooks & Adrian Carter Nature Biotechnology (2000) 18: 1151-1155.) (For details of the latter, see Non-Patent Document 1: Institute of Agricultural Sciences, Ishikawa Agricultural College, Annual Report, 2000, No. 9, 2000, pp. 16-18 (published on Oct. 25, 2001), and Non-Patent Document 2: Institute of Agricultural Sciences, Ishikawa Agricultural College, Annual Report, 2001, No. 10, 2001, pp. 13-16 (published on Sep. 25, 2002), and Non-Patent Document 9: Pogue G P, Lindbo J A, Garger S J, Fitzmaurice W P Making an ally from an enemy: Plant virology and the new agriculture. Annu Rev Phytopathol. 2002, 40: 45-74.)
Previously, the inventors of the present invention have constructed a gene expression system (hereinafter referred to as “high-level mRNA induction and amplification system”). In this system, a replicase gene of a plant virus (brome mosaic virus) and a useful protein gene amplified by the replicase gene are incorporated in the plant chromosomes, and the expression of the replicase gene is controlled for the synthesis of a useful protein. The high-level mRNA induction and amplification system was used in Nicotiana benthamiana plants, and expression of one of the subunits of the replicase, 1a protein, was induced by the steroid hormone control system. This enabled amplification of the gamma interferon gene at RNA level (see Non-Patent Documents 1 and 2).
Further, the inventors of the present invention constructed a steroid hormone-induced high-level mRNA amplification system for foreign proteins, in which tomato mosaic virus (ToMV), a member of the genus Tobamo mosaic virus, was used as a vector. ToMV is a highly replicative single strand RNA virus with a suppressor against a virus resistance reaction (silencing reaction). This system was used in Nicotiana benthamiana plants, and induced expression was attempted under the steroid hormone control system, using a green fluorescent protein gene (hereinafter “GFP gene”) as a reporter gene. The result confirmed amplification of the gene at RNA level, as well as GFP expression (see Non-Patent Document 2).
Meanwhile, there have been attempts to produce useful proteins using plant culture cells. For example, there has been a report that a recombinant protein is produced in a tobacco BY-2 cell using the cauliflower mosaic virus 35S promoter (Non-Patent Document 3: Matsumoto S, Ikura K, Ueda M, Sasaki R. “Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells.” Plant Mol boil. 1995 March; 27(6): 1163-72). In another example, protoplast tobacco BY-2 cells are inoculated with a variant virus RNA vector in which a target peptide gene has been ligated to the 3′ end of a coat protein gene of tobacco mosaic virus (hereinafter “TMV”), so as to cause expression of a fusion protein fused with the coat protein (Non-Patent Document 4: Takamatsu N, Watanabe Y, Yanagi H, Meshi T, Shiba T, Okada Y. “Production of enkephalin in tobacco protoplasts using tobacco mosaic virus RNA vector.” FEBS Lett. 1990 Aug. 20; 269(1): 73-6).
Among the foregoing protein producing methods in plants, the method using transgenic plants enables protein production by simple cultivation of plants. However, a problem of this method is that productivity of each cell is considerably poor. On the other hand, the method in which a plant is infected with a virus vector has good productivity but the method suffers from poor operability due to the inoculation procedure it requires. Another problem of the method is that mass production is difficult due to safety reasons, which includes viral spreading.
Referring to the high-level mRNA induction and amplification system constructed by the inventors with the brome mosaic virus, the brome mosaic virus does not have a suppressor against the virus resistance reaction (silencing reaction). Thus, when a recombinant virus is used that cannot form particles, the viral RNA is degraded by the silencing reaction (virus resistance reaction). That is, while the high-level mRNA induction and amplification system using brome mosaic virus allows a coding gene of the target protein to be amplified at RNA level, it cannot sustain high-level production of protein because the RNA is degraded overtime by the virus resistance reaction (silencing reaction). Another drawback of this system is that the transcription factor that was activated by steroid hormone causes etiolation or other undesirable effects, which is detrimental to plant growth.
The problem of RNA degradation can be avoided and the protein can be efficiently produced when the system using ToMV having a suppressor against the virus resistant reaction (silencing reaction) is used to produce protein in a plant. However, the system is associated with various problems: (1) Difficulties in producing protein on a large scale due to system requirements such as facilities for plant cultivation; (2) A relatively long time period for producing plants; (3) Safety problems posed by spreading of seeds or pollens, etc., of the transformed plants; and (4) Complex procedures.
As to the protein producing system using the tobacco BY-2 cells described in Non-Patent Documents 3 and 4, the system allows for protein production on cell culture, and is therefore suited for large-scale production. Further, the system offers fast amplification, which is advantageous when time is of concern. Another advantage is that the system is safe to use, owning to the fact that the cultured cells quickly die off even if the cells leak out of the system. However, methods based on this system still have the problem of poor productivity and the problem of complex procedure (forming protoplasts, inoculation), among others.
The inventors of the present invention have also developed a novel protein synthesis system (high-level mRNA induction and amplification system) that offers large-scale production with good productivity and good safety. A feature of the high-level mRNA induction and amplification system is that a viral replicase gene and a useful protein gene amplified by the replicase gene are incorporated in plant chromosomes, and that expression of the replicase in the recombinant plant is controlled to control synthesis of the useful protein (see Non-Patent Document 6: Mori, M., Fujihara, N., Mise, K. and Furusawa, I. (2001) Inducible high-level mRNA amplification system by viral replicase in transgenic plants. Plant J 27(1), 79-86).
Further, the inventors modified the foregoing high-level mRNA induction and amplification system with the use of a virus that has a suppressor against the silencing reaction of plants (see Non-Patent Document 2).
Meanwhile, the inventors of the present invention introduced a ribozyme sequence at the 3′ end of viral cDNA, and produced a transformant with a tobacco plant. Transcription with the cauliflower mosaic virus 35S promoter enhanced viral RNA amplification as compared with a sample without the ribozyme (Non-Patent Document 7: Kaido, M., Mori, M., Mise, K., Okuno, T. and Furusawa, I. (1997) Auto-cleavable ribozyme sequence attached to brome mosaic virus cDNAs enhances accumulation of viral RNAs transcribed in vivo from the cDNAs. Ann. Phytopathol. Soc. Jpn. 63, 95-98). Further, by an Agrobacterium method, a tobacco plant was transfected with tobacco mosaic virus cDNA that has been appended with a ribozyme sequence at the 3′ end. This almost doubled the infection rate (Non-Patent Document 8: Turpen, T. H., Turpen, A. M., Weinzettl, N., Kumagai, M. H. and Dawson W. O. (1993) Transfection of whole plants from wounds inoculated with Agrobacterium tumefaciens containing cDNA of tobacco mosaic virus. J. Virol. Methods. 42(2-3), 227-239).
In order to produce a useful arbitrary protein in plants, cultured tobacco BY-2 cells were transformed by the modified high-level mRNA induction and amplification system. However, only at most 5% of the cells showed virus amplification. The absence of virus amplification in most cells was considered to be due to a terminator-originating sequence and a poly-A sequence ligated to the 3′ end of viral RNA transcribed from the cDNA in the cell. If this is indeed the case, further improvements need to be made in the high-level mRNA induction and amplification system, by removing the additional sequences attached to the 3′ end of the viral RNA transcribed from the cDNA in the cell.
One way to remove the additional sequences attached to the 3′ end of the viral RNA transcribed from the cDNA in the cell is to use the ribozyme described in Non-Patent Documents 7 and 8. However, Non-Patent Document 7 merely examines the extents of effects exerted by the presence or absence of ribozyme in regard to amplification of viral RNA, and it is not intended to express a foreign protein or use an inducible promoter. As to Non-Patent Document 8, no transformant is produced. Rather, the publication merely describes the result of temporary infection from cDNA using Agrobacterium. The report concludes that the addition of ribozyme almost doubles the infection rate. To this date, it has been believed that the effects of ribozyme sequence is limiting in the virus vector of the tobacco mosaic virus and other members of the genus Tobamovirus, and that it is not effective to add the ribozyme sequence for the purpose of causing protein production in the Tobamovirus vector.
As described above, the inventors of the present invention successfully improved the amplification rate of mRNA in the high-level mRNA induction and amplification system by using the virus that includes a suppressor against the silencing reaction of plants (Non-Patent Document 2 and Non-Patent Document 10: Institute of Agricultural Sciences, Ishikawa Agricultural College, Annual Report, 2002, No. 11 2002, pp. 14-15 (Published on Dec. 26, 2003).
Further, the inventors of the present invention have found that the ribozyme sequence added to the 3′ end of the viral cDNA in the high-level mRNA induction and amplification system significantly enhanced the amplification of viral RNA as compared with the absence of the ribozyme sequence (for example, see Non-Patent Document 10).
A process for producing a foreign protein with the high-level mRNA induction and amplification system developed by the inventors of the present invention is excellent means for efficiently, inexpensively, and safely producing foreign proteins.
However, in producing (obtaining) transformants (cells), there are often cases where the expression level of virus vector and foreign protein differs between different lines of the resulting transformants (cells). Such a difference in expression level was considered to be due to the positions on the chromosome where the coding gene of the transcription factor and the coding gene of the virus vector are incorporated. More specifically, in the conventional virus vector transfer methods, a coding gene of the virus vector and a coding gene of the transcription factor are transferred into a host cell by being ligated to each other on the same vector. As such, these genes are incorporated in the same position of the chromosome.
However, it is not necessarily the case that the chromosomal locus suitable for the expression of the virus vector is also suitable for the expression of the transcription factor. For example, the genes may be incorporated in the chromosomal locus suitable for the expression of the virus vector but this particular locus may not be suitable for the expression of the transcription factor, and vice versa. In this case, the efficiency of induced expression conferred by the virus vector is low even if the cells have been transformed, and accordingly the expression level of foreign protein is also low.
Further, with current techniques, one must rely on chances as to the transfer position of the vector on the chromosome. For this reason, the probability that transformants (cells) expressing the virus vector and the target protein at high efficiency are obtained is considerably low, and large numbers of transformants (cells) need to be screened for a desirable transformant (cell) line. Thus, protein production using the high-level mRNA amplification system requires a large amount of time and labor.